RNA interference (RNAi) is a mechanism of inhibiting the expression of a specific gene (ie gene silencing), wherein double-stranded RNA molecules induce sequence-specific degradation of the mRNA transcripts of a given gene, thereby inhibiting translation of the mRNA into protein. The RNAi pathway is conserved within plants and mammalian cells (Fire et al., 1998) and is thought to be important for inhibiting viral replication, regulating development, and maintenance of the genome.
Is thought that when exogenous double-stranded RNA (eg a virus with an RNA genome) is encountered by a cell, the RNA is imported into the cytoplasm and cleaved into short fragments by an enzyme termed Dicer. These short fragments are 21- to 25-nucleotide, double-stranded small interfering RNA (siRNA) molecules with 2-nucleotide overhangs at the 3′ ends. The siRNA molecules are then believed to be incorporated into a complex termed the RNA-induced silencing complex (RISC; Hannon and Rossi, 2004). Here, the two strands of the siRNA molecule are separated, with one strand (the guide strand) retained by the complex, while the remaining strand (the passenger strand) is degraded. Either strand can be the guide strand, and factors such as the thermodynamic stability of the 5′ ends of the duplexes can enhance the likelihood of a given strand being selected as the guide strand (Khvorova et al., 2003). The guide strand actively “guides” the activated RISC to complementary (sense) mRNA sequences, triggering cleavage of the mRNA sequence by an Argonaute protein (Diederichs and Haber, 2007), which ultimately prevents or reduces translation of the mRNA into protein, thereby “silencing” or “knocking down” gene expression. siRNA molecules are generally considered to be perfectly complementary; that is, they perfectly base-pair bind to their complementary nucleotide sequence following Watson-Crick base pairing for RNA sequences, such that adenine (A) bases pair with uracil (U) bases, and cytosine (C) bases pair with guanine (G) bases.
Synthetically synthesised siRNA molecules introduced into cells can enter this RNAi pathway and have become an important research tool for knocking down the expression of a specific gene of interest in the laboratory to investigate its function. Such molecules also offer considerable promise for treating diseases associated with the inappropriate expression (eg over-expression) of a particular protein.
During attempts to develop techniques to efficiently deliver synthetic siRNA molecules and artificially trigger RNAi in vivo, it has been noted that siRNA molecules could activate cells of the immune system and induce the production of cytokines both in vivo and in vitro (Sledz et al., 2003; Marques and Williams, 2005; Sioud and Sorensen, 2003; Kariko et al., 2004). Generally, this immunostimulatory effect of siRNA has been regarded as a deleterious, non-specific side-effect that reduces the potential of RNAi to be used both in the laboratory and in the clinic, and substantial research has therefore been undertaken to understand and reduce immunostimulation associated with artificial triggering of RNAi.
In this regard, it has been found that double-stranded RNA molecules stimulate the human innate immune response through toll-like receptor (TLR) 7 and TLR8 in a sequence dependent manner. The relative functional importance of TLR7 and TLR8 is unclear because TLR7 is functional in both humans and mice, whilst TLR8 is functional only in humans (Heil et al., 2004). The binding of ligands to human TLR7 has been shown to stimulate mainly interferon (IFN)-α responses in plasmacytoid dendritic cells (pDC; Hemmi et al., 2002; Jurk et al., 2002), whilst the binding of ligands to human TLR8 has been shown to induce production of proinflammatory cytokines, notably tumour necrosis factor (TNF)-α, in activated monocytes (Gorden et al., 2005; Jurk et al., 2006). It has also been shown that IFNα induction may be more specific to human TLR7 signalling and that TNFα induction may reflect more of a human TLR8 response (Gorden et al., 2005).
Further, it has been shown that the size of siRNA molecules may play a role in immunostimulation, as shorter siRNA molecules have been shown to be poor inducers of IFNα expression in pDCs (Hornung et al., 2005). Moreover, guanine- and uridine-rich sequences seem to be preferentially recognised by the innate immune response system (Heil at al., 2004; Diebold et al. 2004), and “GU” motifs may be involved (Heil et al., 2004); however, some siRNA molecules are, notably, not immunostimulatory despite being guanine- and uridine-rich (Sioud, 2005), and it remains unclear if immunostimulatory motifs are recognised in the context of double-stranded RNA (Marques and Williams, 2005). It is, accordingly, possible that the position of the guanylate and uridylate nucleotides within an siRNA molecule, particular motifs (such as GU motifs) or the proportion of guanylate and uridylate nucleotides may play a role in the nature of the cytokine response induced. However, while many researchers have investigated using siRNA sequences with a low guanylate and uridylate content in order to avoid stimulating an innate immune response, it has been found that it is not always possible to pick a suitable sequence for siRNA molecules (ie one that is low in guanylate and uridylate nucleotides) that still efficiently silences gene expression.
The present applicant has realised that the activation of the innate immune response system when silencing the expression of genes in certain scenarios (eg when treating viral infections and cancerous tumours) is desirable as it may actually enhance the therapeutic effect of treatment, as immunostimulation may enhance anti-viral or anti-tumour immune responses. Further, the present applicant has designed a modification that may be potentially made to any double-stranded siRNA molecule, which enhances or confers immunostimulatory activity while maintaining a desired gene silencing effect.